T cells from NFAT1-/- mice show a biased toward Th2 differentiation and express profuse amounts of IL-4 and Th2 cells, a defect rising from an inability to downregulate IL-4. To investigate other cytokines that may be aberrantly regulated in absence of NFAT1, Dr. Rao's laboratory crossed NFAT1 negative mice with IL-4 negative mice. The absence of IL-4 permits study of the expression of other cytokines without the masking effects of IL-4, e.g., suppression of IFN-gamma. T cells from NFAT1-/- IL-4-/- mice stimulated with anti-CD3 mAb secrete wildtype levels of IL-2, IL-3, and GM-CSF; however, they expressed 8-10 fold less IFN-gamma than T cells from IL-4-/- mice. This IFN-gamma defect was observed when naive T cells or differentiated Th1 cells were stimulated with anti-CD3. Addition of IL-12 elevated synthesis of IFN-gamma, but again not to the levels observed with IL-4-/- mice. Lastly, mixing experiments with wildtype T cells did not reconstitute the mutants' cell production of IFN-gamma. These results suggest two possibilities for the reduction in synthesis (not mutually exclusive). First, NFAT1 binding to regulatory elements in the IFN-gamma locus may be required for optimal IFN-gamma transcription in differentiated Th1 cells that have been acutely stimulated in a manner that activates NFAT1. In support, stimulation with anti-CD3 mAb in inhibitable by the NFAT inhibitor CsA. Second, NFAT1 may be required for a developmental step in the generation of IFN-gamma producing T cells, or at the stage of chromatin remodeling by naive T cells during their differentiation into mature Th1 cells. In support, combined stimulation with cytokines IL-12 and IL-18, two strong NFAT-independent activators of IFN-gamma synthesis, yield reduced levels of IFN-gamma in NFAT1 negative T cells. In this proposal, I will specifically test these two hypotheses to understand the role of NFAT1 in IFN-gamma gene transcription. In Aim 1, I will reconstitute NFAT1-/- IL-4-/- T cells with NFAT1 retrovirus and examine whether successful reconstitution correlates with increased IFN-gamma production by the NFAT1- expressing cells. In Aim 2, I will define the region of the human IFN-gamma gene required for responsiveness to NFAT1, CsA, IL-12 and IL-18, and downregulation by IL-4. In Aim 3, I will map constitutive and inducible DNAse1 hypersensitivity sites in the IFN-gamma locus in IL-4-/- and NFAT1-/- IL-4-/- T cells, to identify regulatory regions whose function may depend upon the presence of NFAT1. In Aim 4, I will map inducible hypersensitive sites to within approximately 500 bp, test whether they can function as inducible enhancer elements in reporter assays, determine whether they bind NFAT1 and possibly other tissue- specific and Th subset-specific transcription factors in vitro, and ask whether binding of these transcription factors is important for enhancer function.